Heartburn is the main symptom of gastroesophageal reflux disease (GERD) which accounts for > 5.5 million office visits/year and its burden is >$9 billion/year in the United States. While in a majority of GERD patients, acid suppression is an effective therapy for this pain, in fully 20-30% of patients? heartburn is not relieved; these people often resort to centrally acting drugs for relief. Our long-term goal is to fill a major gap in the understanding of the afferent pathways mediating heartburn to enable novel strategies to relieve acid suppression-resistant pain. We will address our novel hypothesis that heartburn is mediated by a relatively unstudied nociceptor subtype, namely the vagal jugular afferent C-fibers. Our hypothesis is based on two premises: Firstly, there are two distinguishable but overlapping types of painful esophageal sensations in humans: the heartburn, which is reliably evoked by esophageal acid infusion, and esophageal pain (more similar to other types of visceral pain), which is reliably evoked by esophageal distention. Simultaneously, there are two subtypes of of esophageal C- fiber nociceptors (vagal jugular and spinal DRG) with afferent properties that favor preferential detection of acid (vagal jugular nociceptors innervating the mucosa) and mechanical distention (distention-sensitive DRG nociceptors). In Aim 1 (discovery of novel target) we will determine which vagal afferent nerve subtype(s) has peripheral nerve terminals in the esophageal mucosa such that it is likely to be exposed to luminal acid, is activated by acid, and has central terminals in the brainstem areas implicated in perceptions of visceral noxious stimuli. Vagal esophageal afferent nerves can be divided into 4 major subtypes: P2X2+/TRPV1- nodose mechanoreceptors, P2X2+/TRPV1+ nodose C-fibers acid-, Tac1+/TRPV1+ jugular nociceptors, and poorly characterized capsaicin-insensitive jugular fibers Tac1+/TRPV1-. We have developed innovative approaches to selectively express reporters in these subtypes including TRPV1-Flp crossed with P2X2-Cre or Tac1-Cre to obtained mice in which nodose or jugular subtypes can be selectively targeted by appropriate dual Flp-/Cre- sensitive AAV-vectors. In Aim 2 (validation of novel target) we will begin to evaluate the role of selective activation of only vagal jugular C-fibers (Tac1+/TRPV1+ subtype) in initiating painful sensations from the esophagus in the mouse and rat by selectively expressing the DREADD receptor hM3Dq in esophageal jugular C-fiber (Tac1+/TRPV1+) neurons and evaluating the pain-induced responses to systemic administration of hM3Dq agonist clozapine N -oxide (CNO).